Search Results (111)

Correlative microscopy techniques are used for many different applications in the biological sciences because the comparison of different imaging methods allows researchers to gain more and often complementary information about their samples. Correlative light and electron microscopy (CLEM) methods have been developed to preserve biological samples to withstand the harsh environments necessary for electron microscopy. After first being imaged using widefield (WF) and super-resolution structured illumination fluorescence microscopy (SIM), a NanoSuit chemical treatment was applied to a mammalian tissue sample before imaging with scanning electron microscopy (SEM). This was done to compare the image quality and resolution of each technique. SEM yields higher resolution and offers validation of results from SIM. Full article

In recent years, hybrid materials have been widely applied in the cultural heritage conservation field, especially to preserve color pigments. Among these, one of the most problematic (in terms of conservation science) is the red pigment cinnabar/vermilion. The challenge of this work was to prepare a hybrid coating consisting of a fluorinated polymer (known to protect cinnabar/vermilion), further modified with an inorganic filler based on anatase TiO₂. The latter is suitable because it is functionalized with quenchers, the particles are well above the nanoscale (≥200 nm in diameter), and it was added to the polymer matrix in small quantities. These characteristics made it suitable as a hybrid coating for protecting natural cinnabar, as demonstrated by the results obtained through a multi-analytical approach, based on multispectral imaging, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) coupled with energy-dispersive X-ray analysis (EDX), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), X-ray fluorescence (XRF), contact angle, spectrophotometry and mechanical tests, which were applied to evaluate the performances of the hybrid coating on laboratory specimens (after aging) and original samples. The experimental results provide insight into both the physicochemical decomposition mechanism of natural cinnabar under laboratory-simulated aging conditions and the benefits of the coating. In particular, the treatment did not induce electrochemical changes in the mercury, which remained in its oxidized state (+2) rather than being further reduced to elemental mercury (Hg⁰), the species responsible for the blackening of cinnabar/vermilion (also combined with meta-cinnabar). In the oxidized form (Hg²⁺), the protein binder was altered, yet the application of the hybrid coating did not cause further physicochemical changes (i.e., red shift) to the Hg²⁺/egg-based binder system. This was also reflected in the color properties, which underwent no significant alteration. Finally, the mechanical tests yielded satisfactory results, particularly regarding water vapor permeability and treatment efficiency (even eight months after the initial application, although studies on the same samples are still ongoing). The hybrid coating was ultimately applied to original samples collected at Poggio Spaccasasso (Tuscany, Italy), which could be representative of prehistoric artworks based on natural cinnabar and traces of prehistoric adhesives made from beeswax, natural oils, and plant resins. Full article

Single-molecule Förster resonance energy transfer (smFRET) enables direct measurement of nanoscale conformational dynamics and heterogeneity in biomolecules, but quantitative interpretation of smFRET data critically depends on well-controlled excitation geometry, low background fluorescence, robust calibration, and reproducible data-analysis workflows. Prism-based total internal reflection fluorescence (pTIRF) microscopy provides important advantages for such measurements by physically separating excitation and emission paths and generating a highly confined evanescent field, yet practical guidance for implementing reproducible, quantitative pTIRF systems remains fragmented. Here we present a comprehensive, standardized framework for the design, alignment, calibration, validation, and operation of a prism-based TIRF microscope optimized for single-molecule fluorescence measurements. We describe the complete optical architecture for dual-color excitation and detection, establish alignment invariants that ensure reproducible evanescent excitation and stable donor–acceptor channel registration, and detail surface preparation, flow control, and photostabilization strategies required for reliable long-term imaging. Quantitative benchmarking protocols are introduced to evaluate signal-to-noise ratio, photobleaching kinetics, and spectral crosstalk, providing objective criteria for defining optimal operating conditions and instrument performance limits. Finally, we integrate these experimental procedures with an end-to-end single-molecule data-analysis workflow encompassing channel registration, automated and manual trajectory selection, FRET calculation, and kinetic analysis using hidden Markov modeling. The utility of the platform is demonstrated through smFRET measurements of conformational dynamics in a model nucleic acid system. Together, this work provides a reproducible and accessible methodology for implementing prism-based TIRF microscopy as a robust quantitative platform for single-molecule fluorescence studies across a wide range of biomolecular systems. Full article

This review systematizes the principal methods for imaging and morphological analysis of burn wounds, ranging from light, electron, and fluorescence microscopy to tomographic techniques and mass spectrometry imaging. Light microscopy with histological staining and immunohistochemistry remains the morphological gold standard, enabling visualization of the zones of coagulation, stasis, and hyperemia, as well as molecular characterization of inflammation, angiogenesis, and fibrosis. Electron microscopy allows the study of the ultrastructure of cells and the extracellular matrix at nanometer resolution. Among optical methods, wide-field indocyanine green angiography demonstrates high accuracy in burn depth stratification, whereas fluorescence lifetime imaging microscopy assesses cellular metabolism without exogenous labels. Among tomographic techniques, high-frequency ultrasound is the most accessible bedside modality with submillimeter resolution, permitting evaluation of tissue anatomy, perfusion, and biomechanical properties. magnetic resonance imaging is limited by its high cost and long examination time, while mass spectrometry imaging is used solely for research purposes. For clinical practice, the optimal combination is high-frequency ultrasound and wide-field fluorescence imaging. All methods retain high relevance for experimental research, enabling the validation of novel therapeutic strategies. Full article

Computational clearing (CC) enhances widefield (WF) fluorescence microscopy by suppressing out-of-focus haze and autofluorescence, yielding semi-confocal quality images suitable for segmentation and image-based phenotyping. Here, we propose an “image tracing” workflow for inflammatory mouse intestinal organoids (mIOs) using paired CC and WF images to generate a differential signal (CC − WF). mIOs were derived from intestinal crypts of Lgr5-EGFP stem cell reporter mice and expanded under epidermal growth factor, Noggin, and R-spondin (ENR) conditions. Inflammation was induced by dextran sulfate sodium (DSS) treatment. CC processing enhanced phalloidin-stained apical F-actin and improved EGFP signals by reducing background noise, enabling robust segmentation and quantitative extraction of image morphometrics including area, circularity, and perimeter. CC-WF vectors derived from three-dimensional area–perimeter–circularity plots sensitively captured DSS-induced epithelial disruption analogous to a leaky-epithelium phenotype. Transcriptomic analysis by RNA-seq of DSS-treated mIOs revealed upregulation of inflammatory pathways including TNF-α signaling via NF-κB and IL-6/JAK/STAT3, aligning with microscopy findings. In a proof-of-concept demonstration using phalloidin-stained fluorescence images, ROC analysis of the CC-WF workflow achieved an AUC = 0.95 with 87.5% sensitivity and 92.9% specificity in distinguishing intact from injured mIOs. Full article

Background: Eyelid malignancies require accurate intraoperative margin control to achieve complete tumor excision while preserving the functional and aesthetic integrity of the periocular region. Mohs micrographic surgery (MMS) is widely regarded as the reference standard for margin-controlled excision, whereas frozen section–controlled excision (FSC) represents a reliable and widely used alternative in oculoplastic practice. In parallel, several emerging imaging technologies are being investigated to improve real-time tumor detection and surgical precision. Methods: A narrative review of the literature was conducted to summarize current evidence on intraoperative margin control in eyelid tumor surgery. The review focused on established surgical techniques, including MMS and FSC, as well as emerging imaging modalities such as fluorescence confocal microscopy, reflectance confocal microscopy, optical coherence tomography, line-field confocal optical coherence tomography, photoacoustic imaging, and artificial intelligence (AI)-assisted analysis. Results: MMS provides complete circumferential peripheral and deep margin assessment and remains the benchmark for high-risk, recurrent, and poorly defined periocular tumors, particularly basal cell carcinoma. FSC offers favorable oncologic outcomes, allows immediate reconstruction, and remains an effective option when MMS is not available. Emerging imaging modalities have shown promising diagnostic performance for tumor detection, presurgical mapping, and intraoperative support, particularly in basal cell carcinoma, although evidence in periocular tumors remains limited for most techniques. AI-assisted approaches have also demonstrated high accuracy in the interpretation of frozen sections and optical imaging data, suggesting potential to improve workflow efficiency and diagnostic consistency. Conclusions: MMS and FSC remain the current standards for intraoperative margin control in eyelid tumor surgery. Emerging imaging technologies and AI-based tools may further enhance surgical precision and tissue preservation, but most remain investigational in the periocular setting. Further prospective studies are needed to validate their clinical utility, define standardized workflows, and clarify their role alongside established histopathologic techniques. Full article

Pulsed electric fields (PEFs) are widely recognized as a non-thermal, selective physical therapy with wide clinical application in tumor ablation. The pulse width determines how electrical energy is distributed across plasma membrane to intracellular organelles. However, under an engineering-defined equal-dose condition (N·E²·tp), which serves as a practical control parameter rather than a measure of true cellular energy absorption, systematic and comparable experimental characterization of cellular and subcellular responses across pulse widths from the microsecond to nanosecond range remains limited. In this study, PEFs with pulse widths ranging from 100 μs to 50 ns were applied under equal-dose constraints, and cellular responses were evaluated using transmission electron microscopy (TEM), multi-organelle fluorescence imaging, and flow cytometry. The results indicate that pulse-width-dependent effects were observed under a fixed pulse-number, dose-equalized framework in which electric field strength varied across conditions. Structural and functional changes were observed in multiple organelles, including the nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus. Notably, nanosecond pulses were more effective in inducing mitochondrial membrane potential loss and increasing the proportion of apoptotic or non-viable cells. These findings demonstrate that, under equal-dose conditions, pulse width is a key temporal parameter governing PEF-induced biological effects, indicating that identical dose constraints do not necessarily result in equivalent biological responses. This work provides experimental foundation for parameter selection and optimization in PEF-based biomedical applications. Full article

Background/Objectives: Transdermal formulations are widely utilized in the pharmaceutical and cosmetic fields because they enable non-invasive administration and sustained local drug delivery. Conventional ex vivo skin permeation experiments using Franz diffusion cells have limitations in capturing the spatial and temporal dynamics of skin penetration. This study aimed to develop a confocal laser scanning microscopy (CLSM)-based approach to visualize and semi-quantitatively assess the penetration behavior of fluorescent dyes with differing lipophilicities. Methods: Four fluorescent dyes with different Log P values—Rhodamine B (Rho-B), Rhodamine 123 (Rho-123), Fluorescein Sodium (Flu-Na), and Nile Red (NR)—were formulated into lotion-based vehicles and applied to excised human abdominal skin. CLSM imaging was performed from 10 min to 240 min post-application. Fluorescence intensities were extracted from depth-resolved regions (R1–R4, 30-μm intervals) to examine penetration kinetics and distribution. Results: CLSM imaging demonstrated that Rho-B penetrated through stratum corneum and entered deep into the skin via the hair follicles. Rho-123 and Flu-Na exhibited intercellular and follicular penetration; however, Flu-Na showed only a slight increase in intensity over time; NR showed negligible penetration into the deeper layers. The results of our analysis indicated that moderately lipophilic substances such as Rho-B and Rho-123 diffused deeply into the skin via both transdermal and follicular routes, whereas highly hydrophobic or lipophilic substances remained in the superficial layers. Conclusions: The CLSM-based approach enabled spatially and temporally resolved, semi-quantitative evaluation of transdermal penetration in a single, non-destructive experiment. Although restricted to fluorescent probes, this approach provides a practical early-stage screening tool for comparing route-specific and time-dependent penetration behaviors of compounds with different lipophilicities. Full article

Back focal plane (BFP) imaging has emerged as a widely used technique for investigating various nanoscale optical devices. The ability to provide the full angular distribution of emitted light has enabled the engineering of precise radiation patterns, enabling new advances in nanophotonics. Continuous improvements in the BFP imaging technique, including wavelength, polarization, and phase-resolved signal detection, have allowed us to gain crucial insights into the various optical and material properties of nanophotonic devices. In this study, we introduce a fluorescence lifetime-resolved BFP imaging configuration, which uses a spatial filtering technique in the Fourier plane to discriminate between different emission directions. Uniform silver film (45 nm) with a PMMA matrix layer of about 20 nm containing Rhodamine 6G fluorescent molecular dye was prepared and measured using total internal reflection ellipsometry (TIRE). A coupled oscillator model was used, and strong coupling was observed with a coupling strength of 160 meV. Time-correlated single-photon counting was used for the estimation of fluorescence lifetime in the sub-nanosecond regime, and a direction-dependent lifetime was observed in the BFP imaging configuration. This modified fluorescence-lifetime-resolved BFP microscopy method is essential for directly correlating the collective quantum dynamics (lifetime/decay rate) with the far-field radiation pattern (angle/coherence). It offers a critical tool for designing and optimizing quantum nanophotonic devices, such as polariton-based components and highly directional single-photon emitters, where controlling both excited-state dynamics and spatial coherence is paramount. Full article

Nearly four decades have passed since fluorescence in situ hybridisation was first applied in plants to support molecular cytogenetic analyses across a wide range of species. Subsequent advances in DNA sequencing, bioinformatic analysis, and microscopy, together with the immunolocalisation of various nuclear components, have provided unprecedented insights into the cytomolecular organisation of the nuclear genome in both model and non-model plants, with crop species being perhaps the most significant. The ready availability of sequenced genomes is now facilitating the application of state-of-the-art cytomolecular techniques across diverse plant species. However, these same advances in genomics also pose a challenge to the future of plant molecular cytogenetics, as DNA sequence analysis is increasingly perceived as offering comparable insights into genome organisation. This perception persists despite the continued relevance of FISH-based approaches for the physical anchoring of genome assemblies to chromosomes. Furthermore, cytogenetic approaches cannot currently rival purely genomic methods in terms of throughput, standardisation, and automation. This review highlights the latest key topics in plant cytomolecular research, with particular emphasis on chromosome identification and karyotype evolution, chromatin and interphase nuclear organisation, chromosome structure, hybridisation and polyploidy, and cytogenetics-assisted crop improvement. In doing so, it underscores the distinctive contributions that cytogenetic techniques continue to offer in genomic research. Additionally, we critically assess future directions and emerging opportunities in the field, including those related to CRISPR/Cas-based live-cell imaging and chromosome engineering, as well as AI-assisted image analysis and karyotyping. Full article

Direct stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remains challenging, despite its advantage of requiring only minimal modifications to the imaging setup and sample preparation compared to conventional fluorescence microscopy. A recent advancement that integrates expansion microscopy (ExM), which embeds specimens in a swellable polymer gel, with dSTORM holds promise for attaining imaging resolutions below 10 nm. The combined resolution, however, is governed by the expansion factor of samples, and prior studies have primarily focused on integrations involving approximately 4-fold gel expansion, as dSTORM imaging of high-fold-expanded specimens is still technically demanding. Here, we propose a pragmatic expansion strategy—post-labeling ten-fold robust expansion microscopy (plTREx)—and outline a workflow to facilitate its compatibility with dSTORM, collectively termed plTREx-dSTORM. Specifically, this workflow enhances the mechanical stability of the expansion hydrogel and improves fluorescence signal density across both widefield and dSTORM imaging platforms. Furthermore, we optimize the re-embedding protocol to integrate hydrogel expansion with dSTORM while preventing gel shrinkage. Together, plTREx-dSTORM enables highly refined imaging capable of ultrastructural interpretation of cellular proteins, effectively bridging the resolution gap between electron microscopy and optical microscopy. Full article

Rare earth elements (REEs) are important strategic resources, widely used in various technological fields, especially heavy rare earth elements (HREEs). China has extensive rare earth deposits, with diverse mineral types and a complete range of rare earth elements, characterized by a “heavy south, light north” resource distribution pattern. The rare earth-bearing collophane in the Zhijin area of Guizhou is a typical marine sedimentary phosphorite deposit with large reserves and a high heavy rare earth content. This study investigates the rare earth-bearing collophane in the Zhijin area using X-ray diffraction (XRD), X-ray fluorescence (XRF), and scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS) to analyze its mineral composition and occurrence characteristics. In terms of flotation, a reverse flotation process for magnesium removal was adopted. By optimizing the flotation parameters, including grinding fineness, collector dosage, pH regulator dosage, and depressant dosage, the optimal flotation conditions were determined. A further mineralogical analysis was conducted on both the flotation concentrate and tailings. The results show that the main minerals in the rare earth-bearing collophane of Zhijin are fluorapatite and dolomite, with dolomite as the primary gangue mineral, and rare earth elements are mainly hosted in fluorapatite. The optimal flotation conditions were achieved when the grinding fineness was −74 μm with an 83% passing rate, XF-1 was used as the collector at a dosage of 300 g/t, sulfuric acid (H₂SO₄) as the pH regulator at 6 kg/t, and phosphoric acid (H₃PO₄) as the depressant at 3 kg/t. By employing an optimal reagent regime and implementing a reverse flotation process consisting of one roughing and one scavenging stage, a phosphate concentrate was obtained with a P₂O₅ grade of 31.61% and an REO content of 0.161%. The P₂O₅ recovery reached 84.22%, while the REO recovery was 78.65%. Compared to the raw ore, the P₂O₅ grade increased by 11.52 percentage points, and the REO content improved by 0.051 percentage points. Mineralogical analysis of the flotation concentrate and tailings revealed that dolomite was effectively removed by reverse flotation, while rare earth elements were successfully enriched in the phosphate concentrate. In conclusion, this study provides an efficient flotation separation process for rare earth-bearing collophane and dolomite, while also offering technical support for the efficient recovery of rare earth resources. This research has significant theoretical and practical implications. Full article

Stimuli-responsive polymeric nanostructures are compelling vectors for a wide range of application opportunities. The objective we sought was to broaden the array of self-assembling amphiphilic copolymers with stimuli-responsive characteristics by introducing a hydrophilic tunable monomer, (2-dimethylamino)ethyl methacrylate (DMAEMA), together with a hydrophilic one, lauryl methacrylate (LMA), within linear and branched copolymer topologies. Size exclusion chromatography was used to evaluate the resultant linear and hyperbranched copolymers’ molecular weight and dispersity, and FT-IR and ¹H-NMR spectroscopy techniques were used to delineate their chemical structure. The structural changes in the obtained self-organized supramolecular structures were thoroughly investigated using aqueous media with varying pH and salinity by dynamic light scattering (DLS), fluorescence spectroscopy (FS), and transmission electron microscopy (TEM). The nanoscale assemblies formed by the amphiphiles indicate significant potential for applications within the field of nanotechnology. Full article

The dynamic nature of mitochondria makes live cell imaging an important tool in mitochondrial research. Although imaging using fluorescent probes is the golden standard in studying mitochondrial morphology, these probes might introduce aspecific features. In this study, live cell fluorescent imaging was applied to investigate a pearl-necklace-shaped mitochondrial phenotype that arises when mitochondrial fission is restricted. In this fibroblast-specific pearl-necklace phenotype, constricted and expanded mitochondrial regions alternate. Imaging studies revealed that the formation time of this pearl-necklace phenotype differs between laser scanning confocal, widefield and spinning disk confocal microscopy. We found that the phenotype formation correlates with the excitation of the fluorescent probe and is the result of phototoxicity. Interestingly, the phenotype only arises in cells stained with red mitochondrial dyes. Serial section electron tomography of the pearl-necklace mitochondria revealed that the mitochondrial membranes remained intact, while the cristae structure was altered. Furthermore, filaments and ER were present at the constricted sites. This study illustrates the importance of considering experimental conditions for live cell imaging to prevent imaging artifacts that can have a major impact on the obtained results. Full article

Motility is a vital aspect of many forms of life, with a wide range of highly conserved as well as highly unique systems adapted to the needs of various organisms and environments. While many motility systems are well studied using structural techniques like X-ray crystallography and electron microscopy, as well as fluorescence microscopy methodologies, it is difficult to directly determine the relationship between the shape and movement of a motility system due to a notable gap in spatiotemporal resolution. Bridging this gap as well as understanding the dynamic molecular movements that underpin motility mechanisms has been challenging. The advent of high-speed atomic force microscopy (HS-AFM) has provided a new window into understanding these nano-scale machines and the dynamic processes underlying motility. In this review, we highlight some of the advances in this field, ranging from reconstituted systems and purified higher-order supramolecular complexes to live cells, in both prokaryotic and eukaryotic contexts. Full article